Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication
journal contributionposted on 17.12.2020, 06:27 by AT Zhang, AR Langley, CP Christov, E Kheir, Thomas ShafeeThomas Shafee, TJ Gardiner, T Krude
Non-coding Y RNAs are required for the initiation of chromosomal DNA replication in mammalian cells. It is unknown how they perform this function or if they associate with a nuclear structure during DNA replication. Here, we investigate the association of Y RNAs with chromatin and their interaction with replication proteins during DNA replication in a human cell-free system. Our results show that fluorescently labelled Y RNAs associate with unreplicated euchromatin in late G1 phase cell nuclei before the initiation of DNA replication. Following initiation, Y RNAs are displaced locally from nascent and replicated DNA present in replication foci. In intact human cells, a substantial fraction of endogenous Y RNAs are associated with G1 phase nuclei, but not with G2 phase nuclei. Y RNAs interact and colocalise with the origin recognition complex (ORC), the pre-replication complex (pre-RC) protein Cdt1, and other proteins implicated in the initiation of DNA replication. These data support a molecular 'catch and release' mechanism for Y RNA function during the initiation of chromosomal DNA replication, which is consistent with Y RNAs acting as replication licensing factors. © 2011. Published by The Company of Biologists Ltd.
We thank Cath Green and Sebastian Klinge for critical reading of this manuscript, Rachael Walker for help with flow cytometry and Stephen P. Jackson, Aloys Schepers, Michael Leffak, Stephen D. Bell, Heinz-Peter Nasheuer, Frank Grosse and Anindya Dutta for reagents. This work was supported by Cancer Research UK (project grant C1471/A8448 and vacation student bursary C1471/A10308) and by the Association for International Cancer Research (AICR Project Grant No: 10-0570). A. R. L. and T.J.G. were supported by Research Studentships from the Medical Research Council (MRC). The University of Cambridge Department of Zoology confocal suite was financed by the Wellcome Trust and the Newton Trust. Deposited in PMC for release after 6 months.
Cancer Research UK | C1471/A8448
Cancer Research UK | C1471/A10308
Association for International Cancer Research (AICR) | 10-0570
Medical Research Council (MRC)
Worldwide Cancer Research | 10-0570
JournalJournal of Cell Science
PublisherThe Company of Biologists
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Science & TechnologyLife Sciences & BiomedicineCell BiologyDNA replicationY RNALicensing factorNon-coding RNARO RIBONUCLEOPROTEIN COMPLEXESBINDING PROTEININTRACELLULAR-LOCALIZATIONNONCODING RNASKUHETEROGENEITYREQUIREMENTAUTOANTIGENCOMPONENTCONTAINSChromatinEuchromatinHumansCell Cycle ProteinsRNA, UntranslatedMicroscopy, ConfocalG1 PhaseDNA ReplicationBase SequenceMolecular Sequence DataOrigin Recognition ComplexDevelopmental Biology